PhoB activation in non-limiting phosphate condition by the maintenance of high polyphosphate levels in the stationary phase inhibits biofilm formation in Escherichia coli.

Polyphosphate (polyP) degradation in Escherichia coli stationary phase triggers biofilm formation via the LuxS quorum sensing system. In media containing excess of phosphate (Pi), high polyP levels are maintained in the stationary phase with the consequent inhibition of biofilm formation. The transcriptional-response regulator PhoB, which is activated under Pi limitation, is involved in the inhibition of biofilm formation in several bacterial species. In the current study, we report, for the first time, we believe that E. coli PhoB can be activated in non-limiting Pi conditions, leading to inhibition of biofilm formation. In fact, PhoB was activated when high polyP levels were maintained in the stationary phase, whereas it remained inactive when the polymer was degraded or absent. PhoB activation was mediated by acetyl phosphate with the consequent repression of biofilm formation owing to the downregulation of c-di-GMP synthesis and the inhibition of autoinducer-2 production. These results allowed us to propose a model showing that PhoB is a component in the signal cascade regulating biofilm formation triggered by fluctuations of polyP levels in E. coli cells during stationary phase.

Environmental phosphate differentially affects virulence phenotypes of uropathogenic Escherichia coli isolates causative of prostatitis.

K-12 Escherichia coli cells grown in static media containing a critical phosphate (Pi) concentration ≥25 mM maintained a high polyphosphate (polyP) level in stationary phase, impairing biofilm formation, a phenomenon that is triggered by polyP degradation. Pi concentration in human urine fluctuates according to health state. Here, the influence of environmental Pi concentration on the occurrence of virulence traits in uropathogenic E. coli (UPEC) isolated from acute prostatitis patients was evaluated. After a first screening, 3 isolates were selected according to differential biofilm formation profiles depending on media Pi concentration. For each isolate, biofilm positive and negative conditions were established. Regardless of the isolate, biofilm formation capacity was accompanied with curli and cellulose production and expression of some key virulence factors associated with adhesion. When the selected isolates were grown in their non-biofilm-forming condition, low concentrations of nalidixic acid and ciprofloxacin induced biofilm formation. Interestingly, similar to laboratory strains, polyP degradation induced biofilm formation in the selected isolates. Data demonstrated the complexity of UPEC responses to environmental Pi and the importance of polyP metabolism in the virulence of clinical isolates.

The antimicrobial peptide microcin J25 stabilizes the gel phase of bacterial model membranes.

The bacterial membrane interaction of the antimicrobial peptide microcin J25 was studied with the probe-free techniques Langmuir monolayers and infrared spectroscopy. Membrane model systems composed by phosphatidylethanolamine:phosphatidylglycerol 7:3, which mimic the cytoplasmic membrane of Gram negative bacteria, were used in both monolayer and bilayer approaches. The peptide reduced the transition surface pressure of the expanded-to-condensed lipid monolayer states, as well as increased the gel-to-liquid crystalline transition temperature in bilayers, indicating a stabilization of membrane ordered state. In addition, a reduction of the surface pressure at which condensed domains appeared was observed upon mixed monolayers compression after microcin J25 adsorption. The results indicate a favorable interaction of microcin J25 with bacterial membrane model systems. Also, the effects on the ordered phases stabilization are discussed in terms of the biological effects observed in membranes of sensitive cells.

The antibacterial action of microcin J25: evidence for disruption of cytoplasmic membrane energization in Salmonella newport.

Microcin J25 (MccJ25) is a cyclic peptide of 21 unmodified amino acid residues produced by a fecal strain of Escherichia coli. It has previously been shown that the antibiotic activity of this peptide is mainly directed to Enterobacteriaceae, including several pathogenic E. coli, Salmonella and Shigella strains. In this paper we show that MccJ25 acts on the cytoplasmic membrane of Salmonella newport cells producing alteration of membrane permeability, and the subsequent gradient dissipation, that initiate the inhibition of process, such as oxygen consumption. These results, taken together with our in vitro observations [Rintoul et al. (2000) Biochim. Biophys. Acta 1509, 65-72], strongly suggest that the disruption of the cytoplasmic membrane gradient is closely related to the bactericidal activity of MccJ25 in S. newport.

Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25.

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

Effects of the antibiotic peptide microcin J25 on liposomes: role of acyl chain length and negatively charged phospholipid.

This paper reports the effects of microcin J25 (MccJ25) on the microviscosity and permeability of phospholipid vesicles of different compositions. The results obtained indicate that MccJ25 interacts with egg L-alpha-phosphatidylcholine (PC) vesicles as demonstrated by peptide intrinsic fluorescence determinations. The interaction depends on the lipid composition of the vesicles. MccJ25 interaction induces a significant fluidity increase of egg PC vesicles. This effect is time and concentration dependent. Both trimethyl ammonium 1,6-diphenyl-1,3,5-hexatriene and 1,6-diphenyl-1, 3,5-hexatriene gave the same results. The microviscosity of L-alpha-phosphatidylcholine dipalmitoyl small unilamellar vesicles (SUVs) was affected while that of L-alpha-phosphatidylcholine dimyristoyl vesicles was not, indicating that the effect was strongly dependent on the chain length of fatty acids. On the other hand, negatively charged L-alpha-phosphatidyl-DL-glycerol (PG) vesicles remarkably inhibited the peptide effect. Nevertheless vesicles composed of L-alpha-phosphatidylethanolamine:PG:cardiolipin (7:2:1), a composition resembling bacterial membrane, were sensitive to the MccJ25 effect. MccJ25 effectively dissipated the valinomycin-induced membrane potential, but induced only a modest leakage (5%) of the trapped Tb(+3)-dipicolinic acid complex. These results indicate that the peptides interact and perturb the bilayer of SUVs. The relationships between this effect and bactericidal action remain to be elucidated.

Differential transmembrane diffusion of triiodothyronine and thyroxine in liposomes: regulation by lipid composition.

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3'triiodo l-thyronine and 3,5,3',5' tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2, 4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (125I) 3,5,3'triiodo l-thyronine and (125I) 3,5,3',5' tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3' triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3', 5' tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer.

Differential effect of triiodothyronine and thyroxine on liposomes containing cholesterol: physiological speculations.

The effect of thyroid hormones on the steady-state fluorescence polarization and on the release of the liposomal content was analyzed in liposomes composed of egg phosphatidylcholine and egg phosphatidyl choline:cholesterol in different molar ratios. Depending on liposome cholesterol composition, a dual effect of triiodothyronine was found. The fluorescence polarization of 1,6 diphenyl 1,3,5 hexatriene or 1-(4-trimethylaminophenyl) 6 phenyl-1, 3, 5 hexatriene decreased by the addition of the hormone when cholesterol content was in the range from 0 to 30 moles %, while it increased with cholesterol from 30 to 50 moles %. In the release experiments, the effect of triiodothyronine was also biphasic; the leakage was the highest at 0% and 50% and the lowest at 30 moles % of cholesterol. On the contrary, thyroxine was without effect on liposomes containing cholesterol from 30 to 50 mol %. This fact correlated with a lower incorporation of thyroxine, compared with that of triiodothyronine in liposomes containing up to 30 moles % of cholesterol. The fact that the above differential incorporation of thyroid hormones was also observed at physiological concentration and that most of the mammalian membrane cells have more than 25 moles % of cholesterol have for physiological implications to the observations reported here.

Differential effect of triiodothyronine and thyroxine on the liposomal membrane in liquid-crystalline and gel state.

The effect of thyroid hormones on the degree of order or fluidity of dimyristoyl, dipalmitoyl or egg yolk phosphatidyl choline liposomes was evaluated by fluorescence spectroscopy methods. The freedom of molecular motion above the phase transition temperature was decreased, while below the transition, the mobility was actually increased by the incorporation of triiodothyronine to liposomes. While thyroxine decreases the fluidity in the liquid crystalline state, it cannot increase the fluidity in the gel state. A differential effect of triiodothyronine and thyroxine on the release of the liposomal content was found, depending on the liquid crystalline or gel state of the liposomes. These facts were correlated with the differential incorporation of the hormones to liposomes above and below the phase transition temperature of dimyristoyl and dipalmitoyl phospholipid choline. In gel state, a low incorporation of thyroxine compared with triiodothyronine was found.

Oral carcinoma in pregnancy.

Oral cancer presenting in pregnancy is a rare combination which, because of current epidemiological changes in the disease, may present more often in the future. Treatment of the condition can be very difficult. Decisions have to be made about terminating the pregnancy or finding a balance between the treatment of the mother and protecting the foetus from the effects of various treatment modalities. This may result in a compromise treatment of the disease. To illustrate the problems, we discuss a case of carcinoma of the tongue presenting in a 23-week pregnant Gravida 1 Para 0 lady, and review the literature.

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y produccíon de etanol a partir de lactosa / Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose

Se realizaron estudios sobre levaduras del género Kluyveromyces (K. fragilis 507, K. lactis 29 y K. lactis 10), que crecen en lactosa como única fuente de carbono, ya que poseen el sistema enzimático para la utilización de este azucar. Se determinó la actividad de ß-galactosidasa en medios con glucosa y con lactosa, en cultivos de estas tres cepas en fase log. Al agregar entre ) y 12% v/v de etanol a células tratadas con tolueno, no se observó inhibición enzimática para la cepa K. lactis 10 que tenía la mayor actividad enzimática (704,4 Unidades). Como existe la posibilidad de utilizar industrialmente suero concentrado 4 veces como sustrato que contenga lactosa, se realizaron ensayos de fermentación a 30 C las tres cepas, en medios que contenían inicialmente 16,5 y 24,5% de lactosa. Al cabo de 48 h la lactosa residual fue prácticamente cero, lográndose concentraciones de etanol directo entre 7,60 y 10,10% v/v. Es de esperar que la velocidad de fermentación de un disacárido como lactosa, esté relacionada con la velocidad de hidrólisis del mismo azúcar, por lo que cepas con una mayor velocidad enzimática hidrolítica debieran poseer mayor velocidad de fermentación. En este caso no se observó tal comportamiento, por cuanto cepas con actividad enzimática tan distinta como K. lactis 10 (704,4 U) y K lactis 29(189,7 U) no presentaron mayores diferencias en la producción de etanol a partir de lactosa

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y produccíon de etanol a partir de lactosa / Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose

Se realizaron estudios sobre levaduras del género Kluyveromyces (K. fragilis 507, K. lactis 29 y K. lactis 10), que crecen en lactosa como única fuente de carbono, ya que poseen el sistema enzimático para la utilización de este azucar. Se determinó la actividad de ß-galactosidasa en medios con glucosa y con lactosa, en cultivos de estas tres cepas en fase log. Al agregar entre ) y 12% v/v de etanol a células tratadas con tolueno, no se observó inhibición enzimática para la cepa K. lactis 10 que tenía la mayor actividad enzimática (704,4 Unidades). Como existe la posibilidad de utilizar industrialmente suero concentrado 4 veces como sustrato que contenga lactosa, se realizaron ensayos de fermentación a 30 C las tres cepas, en medios que contenían inicialmente 16,5 y 24,5% de lactosa. Al cabo de 48 h la lactosa residual fue prácticamente cero, lográndose concentraciones de etanol directo entre 7,60 y 10,10% v/v. Es de esperar que la velocidad de fermentación de un disacárido como lactosa, esté relacionada con la velocidad de hidrólisis del mismo azúcar, por lo que cepas con una mayor velocidad enzimática hidrolítica debieran poseer mayor velocidad de fermentación. En este caso no se observó tal comportamiento, por cuanto cepas con actividad enzimática tan distinta como K. lactis 10 (704,4 U) y K lactis 29(189,7 U) no presentaron mayores diferencias en la producción de etanol a partir de lactosa (AU)

[Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose]. / Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y producción de etanol a partir de lactosa.

We investigated the behavior of yeast of the genus Kluyveromyces (K. fragilis 507, K. lactis 29 and K. lactis 10), which grow on lactose as sole carbon source, since they possess an enzyme system for the utilization of this sugar. We determined the beta-galactosidase activity of these strains, grown in the logarithmic phase in media containing glucose and lactose. On addition of 0 to 12% v/v ethanol to cells treated with toluene, we did not observe inhibition of the enzyme in strain 10 of Kluyveromyces lactis, which showed the greatest activity (704.4 Units). Since there exist the possibility of industrial utilization of concentrated whey (4 times), we performed fermentation tests of the three strains, at 30 C, in media containing initial lactose concentrations of 16.5 and 24.5%. After 48 h the residual lactose concentration was practically zero, and the ethanol concentrations had reached 7.60 and 10.10% v/v. It might be expected that the rate of fermentation of a disaccharide such as lactose would be related to the rate of hydrolysis of the sugar, so that strains having a higher rate of enzymatic hydrolysis should show a higher fermentation rate. However, we did not observe such behavior, as strains of Kluyveromyces having enzymatic activities as different as K. lactis 10 (704.4 U) and K. lactis 29 (189.7 U) did not show any great difference in the production of ethanol from lactose.

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y producción de etanol a partir de lactosa. / [Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose]

We investigated the behavior of yeast of the genus Kluyveromyces (K. fragilis 507, K. lactis 29 and K. lactis 10), which grow on lactose as sole carbon source, since they possess an enzyme system for the utilization of this sugar. We determined the beta-galactosidase activity of these strains, grown in the logarithmic phase in media containing glucose and lactose. On addition of 0 to 12

v/v ethanol to cells treated with toluene, we did not observe inhibition of the enzyme in strain 10 of Kluyveromyces lactis, which showed the greatest activity (704.4 Units). Since there exist the possibility of industrial utilization of concentrated whey (4 times), we performed fermentation tests of the three strains, at 30 C, in media containing initial lactose concentrations of 16.5 and 24.5

. After 48 h the residual lactose concentration was practically zero, and the ethanol concentrations had reached 7.60 and 10.10

v/v. It might be expected that the rate of fermentation of a disaccharide such as lactose would be related to the rate of hydrolysis of the sugar, so that strains having a higher rate of enzymatic hydrolysis should show a higher fermentation rate. However, we did not observe such behavior, as strains of Kluyveromyces having enzymatic activities as different as K. lactis 10 (704.4 U) and K. lactis 29 (189.7 U) did not show any great difference in the production of ethanol from lactose.