Bacteriocin Typing of Vibrio cholerae

1970 ◽  
Vol 1 (3) ◽  
pp. 293-299
Author(s):  
A. N. Chakrabarty ◽  
Sati Adhya ◽  
Jayantisri Basu ◽  
Sujata G. Dastidar
Keyword(s):  
Vibrio Cholerae ◽  
Cold Shock ◽  
Casein Hydrolysate ◽  
Bacteriocin Production ◽  
Buffer Concentration ◽  
Indicator Organisms ◽  
Proteose Peptone ◽  
Bacteriocin Typing ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

Bacteriocins of Vibrio cholerae have been demonstrated against enterobacterial and vibrio indicator organisms by conventional techniques. Abundant bacteriocin production took place on casein hydrolysate-yeast extract, tryptic soy, digest broth, proteose-peptone, and neopeptone agars. Essential factors were a citrate-phosphate buffer concentration of 0.5 to 0.7%, at p H 7.5 to 7.6, and cold shock. Thermal treatment of indicator organisms at 45 C for 12 min increased the percentage of typable strains. The bacteriocins of V. cholerae appeared to be powerful diffusible bactericidal agents. By using 8 indicator strains, 11 bacteriocin types have been recognized among 425 strains, of which 87% are typable at present.

Maintenance of ΔpH by a butanol-tolerant mutant of Clostridium beijerinckii

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 607-613 ◽  
Author(s):  
Fanqiang Wang ◽  
Shelby Kashket ◽  
Eva R. Kashket
Keyword(s):  
Phosphate Buffer ◽  
Sodium Citrate ◽  
Potassium Citrate ◽  
Clostridium Beijerinckii ◽  
Wild Type ◽  
Arginine Residues ◽  
Tolerant Mutant ◽  
Mutant Cells ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane ΔpH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The ΔpH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the ΔpH component of the protonmotive force) and supplemented with glucose and Mg2+. The ΔpH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a ΔpH of 1·4 when butanol was added to a concentration of 1·5 % (w/v), while the wild-type ΔpH was reduced to 0·1. The ΔpH of both strains was completely dissipated with 1·75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the ΔpH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0·8 %) prevented this ΔpH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in ΔpH occurred more slowly than in the mutant, and sparing of the ΔpH by 0·8 % butanol was less pronounced. The authors interpret these data to mean that the ΔpH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.

scholarly journals Cold Shock Response and Major Cold Shock Proteins of Vibrio cholerae

2003 ◽  
Vol 69 (11) ◽  
pp. 6361-6369 ◽  
Author(s):  
Partha Pratim Datta ◽  
Rupak K. Bhadra
Keyword(s):  
Vibrio Cholerae ◽  
Cold Shock ◽  
De Novo ◽  
Alternative Pathway ◽  
Regulation Of Gene Expression ◽  
Inducible Promoter ◽  
Small Chromosome ◽  
Cold Shock Proteins ◽  
Shock Proteins ◽  
Kinetics Of

ABSTRACT When exponentially growing Vibrio cholerae cells were shifted from 37°C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15°C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspAVC) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspAVC and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspAVC and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37°C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5′ untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.

scholarly journals Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

2005 ◽  
Vol 49 (8) ◽  
pp. 3341-3346 ◽  
Author(s):  
D. T. A. te Dorsthorst ◽  
P. E. Verweij ◽  
J. F. G. M. Meis ◽  
J. W. Mouton
Keyword(s):  
Amphotericin B ◽  
Phosphate Buffer ◽  
Vitro Activity ◽  
Nonlinear Relationship ◽  
In Vitro Activity ◽  
The Mean ◽  
Ph Range ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

ABSTRACT In this study, we investigated the pH dependency of the in vitro activities of amphotericin B (AMB) and flucytosine (5FC) against Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, Rhizopus spp., and Scedosporium prolificans in RPMI 1640 buffered with citrate buffer (pH 4.0, 5.0, 5.4, and 6.0), citrate-phosphate buffer (pH 5.4, 6.0, 6.4, and 7.0), and 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 6.4, 7.0, 7.4, and 7.9). For 5FC, no significant differences were found between MICs obtained with the different buffers, while for AMB, significant differences were found. The MICs obtained with citrate-phosphate buffer were approximately 1 twofold-dilution step higher than the MICs obtained with MOPS. We demonstrated that the in vitro activities of AMB and 5FC against yeast and mold isolates were pH dependent. The in vitro activity of AMB decreased when the pH was lowered, while the in vitro activity of 5FC increased. The effect of the pH on the in vitro activities was dependent not only on the antifungal agent tested but also on the microorganism. For AMB, there was a nonlinear relationship (median r 2, 0.864) for Candida spp., C. neoformans, A. fumigatus, and Rhizopus spp. over the pH range tested. The mean MICs ranged from 0.5 to 2.52 μg/ml at pH 7.0 and from 20.16 to 32 μg/ml at pH 5.0. For S. prolificans, there was no relationship. For 5FC, there was a linear relationship for Candida spp. (median r 2, 0.767) and a nonlinear relationship for C. neoformans and A. fumigatus (median r 2, 0.882) over the pH range tested. The mean MIC values ranged from 0.125 to 1,024 μg/ml at pH 7.0 and from 0.02 to 4 μg/ml at pH 5.0. For Rhizopus spp. and S. prolificans, the relationship could not be determined, since the MIC was >1,024 μg/ml over a pH range of 4.0 to 7.9.

scholarly journals Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 377-388 ◽  
Author(s):  
BL Evatt ◽  
J Levin ◽  
KM Algazy
Keyword(s):  
Ammonium Sulfate ◽  
Phosphate Buffer ◽  
Carboxymethyl Cellulose ◽  
Room Temperature ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Elution Volume ◽  
Ammonium Sulfate Saturation ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

Abstract Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate- phosphate buffer) and carboxymethyl cellulose (0/003 M citrate- phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta- globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis- stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.

scholarly journals A new allele of peptidase-B in cattle

1997 ◽  
Vol 20 (1) ◽  
pp. 9-12
Author(s):  
Maria Aparecida Cassiano Lara ◽  
Eucleia Primo B. Contel
Keyword(s):  
Genetic Control ◽  
Phosphate Buffer ◽  
Bos Taurus ◽  
16Th Century ◽  
Buffer System ◽  
Red Cell ◽  
Autosomal Locus ◽  
Native Cattle ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

Electrophoretic analyses of peptidase-B were carried out on red cell hemolysates from Holstein, Mantiqueira and Gyr cattle, using cornstarch, known in Brazil as Penetrose-30. We describe a new peptidase-B allele, denoted Pep-B1, in Mantiqueira cattle, belonging to the Bos taurus group, which are the result of a cross of native cattle of Portuguese origin introduced in Brazil during colonial times (16th century) with Holstein and Caracu cattle. The genetic control of peptidase-B was determined by typing parents and progeny segregating for all three alleles, confirming that peptidase B is controlled by a single autosomal locus with three codominant alleles, denoted Pep-B1, Pep-B2 and Pep-B3 The use of the citrate-phosphate buffer system, at pH 5.9, on 14% gel, under the electrophoretic conditions standardized in this study permitted good visualization of all peptidase-B variants.

Detachment of Pseudomonas fluorescens P26 from Beef Rinsed in Salt and Acid Solutions

1984 ◽  
Vol 47 (7) ◽  
pp. 537-541 ◽  
Author(s):  
C. P. APPL ◽  
R. T. MARSHALL
Keyword(s):  
Pseudomonas Fluorescens ◽  
Phosphate Buffer ◽  
Chloride Salt ◽  
Acid Solutions ◽  
Salt Solutions ◽  
Large Loss ◽  
Bacterial Surfaces ◽  
Citrate Phosphate ◽  
Number Of Cells ◽  
Citrate Phosphate Buffer

The purpose of this study was to test the hypothesis that numbers of bacteria detached from meat could be increased by modification of the ionic environment surrounding both the meat and bacterial surfaces. Of five 0.1 M chloride salt solutions that were used to rinse cells of Pseudomonas fluorescens P26 from meat, KCl removed the highest average number of cells. These numbers were significantly greater than those for NH4Cl and MgCl2. Compared with water, the solution of 0.1 M KCl rinsed three times as many bacteria from cubes of inoculated meat shaken in solution for 1 min and there were one-half as many bacteria recovered from meat rinsed with KCl as from meat rinsed with water. The latter difference was significant, and we conclude that KCl assisted in detachment of the pseudomonads. However, no significant effect of 0.1 M KCl was observed when it was added to water that had been buffered to pH 4 and 5 with citrate-phosphate buffer. Instead, the buffered rinse caused a large loss in viability of the pseudomonads. This large loss in viability may have over-shadowed the smaller effect of KCl.

scholarly journals Comparison of induction of B45 Helicobacter pylori prophage by acid and UV radiation

2013 ◽  
Vol 19 (S4) ◽  
pp. 27-28 ◽  
Author(s):  
A.P. Alves de Matos ◽  
P. Lehours ◽  
A. Timóteo ◽  
M. Roxo-Rosa ◽  
F.F. Vale
Keyword(s):  
Electron Microscopy ◽  
Gastric Mucosa ◽  
Uv Radiation ◽  
Human Stomach ◽  
Lytic Cycle ◽  
Acidic Environment ◽  
H Pylori ◽  
Citrate Phosphate ◽  
Microaerophilic Conditions ◽  
Citrate Phosphate Buffer

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of <4, but growth occurs only at the relatively narrow pH range of 5.5 to 8.0, with optimal growth at neutral pH. Recently we have identified a prophage sequence (prophage phiHP33) in the strain B45, isolated from a patient diagnosed with gastric MALT lymphoma. This prophage revealed to be very difficult to induce. In fact, only few phage particles were observed on electron microscopy micrographs after exposure to UV radiation.In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

Preformed magnesium hydroxide precipitate for second-step concentration of enteroviruses from drinking and surface waters

1982 ◽  
Vol 28 (7) ◽  
pp. 783-787 ◽  
Author(s):  
P. Vilagines ◽  
B. Sarrette ◽  
R. Vilagines
Keyword(s):  
Magnesium Hydroxide ◽  
Surface Waters ◽  
Second Step ◽  
Virus Concentration ◽  
Recovery Efficiency ◽  
Experimental Conditions ◽  
Final Volume ◽  
Hydroxide Precipitate ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

A method is described for the second-step concentration of viruses from large volumes of drinking and surface waters. Seeded viruses present in the first eluate, performed with 50 mM glycine buffer, pH 11.5, were adsorbed on a preformed magnesium hydroxide precipitate. After low-speed centrifugation they were desorbed and ajusted to pH 7 with McIlvaine citrate–phosphate buffer. In these experimental conditions 90% of the viruses present in the 300-mL first eluate were reconcentrated in a final volume of 40 mL. The recovery efficiency was independant of either virus concentration or water quality.

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