Cell-free and cell-bound hemolytic activities of Proteus penneri determined by different Hly determinants

1991 ◽  
Vol 37 (6) ◽  
pp. 419-424 ◽  
Author(s):  
Sławomir Łukomski ◽  
Liliana Serwecińska Antoni Różalski ◽  
Jarosław Dziadek ◽  
Paweł Staczek ◽  
Adam Jaworski
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Hemolytic Activity ◽  
Control Mechanism ◽  
Hybridization Technique ◽  
Colony Hybridization ◽  
Representative Cell ◽  
Hemolysin Gene ◽  
Monospecific Antiserum ◽  
Proteus Penneri

A collection of 45 Proteus penneri strains was characterized with respect to their hemolytic activity and representative cell-free or only cell-bound hemolysin possessing strains were chosen for further study. Extracellular Proteus penneri hemolysin, which was investigated earlier by hybridization, reacted with monospecific antiserum against α-hemolysin of Escherichia coli. In this paper we also show, using the colony hybridization technique, that the α-hemolysin-like determinant is widely distributed among Proteus penneri strains. Because one of the strains tested, which expressed a high activity of cell-bound hemolytic factor, did not carry such a Hly determinant, the presence of a second hemolysin is postulated. We cannot demonstrate any difference in hybridization patterns of α- and β-hemolytic Proteus penneri strains and accumulation of the toxin molecule inside the cells was also not observed. The existence of another control mechanism, external to the hly operon, for hemolysin gene is suggested. Key words: Proteus penneri, hemolytic activity.

scholarly journals Molecular characterization of Listeria monocytogenes isolated from foods

1999 ◽  
Vol 30 (4) ◽  
pp. 356-361 ◽  
Author(s):  
Fabiana Cristina Pimenta ◽  
Sirdéia Maura Perrone Furlanetto ◽  
Leonard W. Mayer ◽  
Jorge Timenetsky ◽  
Manoel Armando Azevedo dos Santos
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Factors ◽  
Hemolytic Activity ◽  
Prototype Strain ◽  
Strain Atcc ◽  
Hybridization Method ◽  
Gene Detection ◽  
Colony Hybridization ◽  
Hemolysin Gene

A total of 30 strains of Listeria monocytogenes isolated from different foods (16 of differents kinds of sausage, 14 cheese,) purchased at groceries of São Paulo City were ribotyped and analysed for the presence and expression of hemolysin gene and production of phosphatidylinositol-specific phosphalipase C - PI-PLC enzyme. The L. monocytogenes strains were differentiated into six ribotype classes. A total of 13 (43.3%) from these strains belong to the same ribotype (ribotype I), and was coincident to the ribotype of the standard L. monocytogenes prototype strain (ATCC-15313). The hemolytic activity was observed in 29 (96.7%) strains when incubated at 37oC, but not at 4oC. The direct colony hybridization method for hemolysin gene detection showed a positive reaction whit all the 30 L. monocytogenes strains, while showed negative reaction with other Listeria spp. The PI-PLC was produced by 27 (90%) of the strains analysed. There was no correlation between the six identified ribotypes and the virulence factors (hemolysin and PI-PLC) studied.

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

1991 ◽  
Vol 37 (5) ◽  
pp. 407-410
Author(s):  
Mônica A. M. Vieira ◽  
Beatriz E. C. Guth ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Sensitivity And Specificity ◽  
Dna Probes ◽  
Type I ◽  
Enteropathogenic Escherichia Coli ◽  
Key Words Dna ◽  
E Coli ◽  
Heat Stable

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like, invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathoghens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots. Key words: DNA probes, stool blots, enteropathogens, diagnosis.

DNA Colony Hybridization Method Using Synthetic Oligonucleotides to Detect Enterotoxigenic Escherichia coli: Collaborative Study

1986 ◽  
Vol 69 (3) ◽  
pp. 531-536
Author(s):  
Walter E Hill ◽  
Barry A Wentz ◽  
William L Payne ◽  
James A Jagow ◽  
Gerald Zon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Collaborative Study ◽  
Dna Probes ◽  
Enterotoxigenic Escherichia Coli ◽  
Hybridization Probe ◽  
Colony Hybridization ◽  
E Coli ◽  
Calf Thymus ◽  
Recombinant Dna Techniques

Abstract The genes that encode several of the enterotoxins produced by Escherichia coli have been cloned by recombinant DNA techniques. When the nucleotide sequence of these genes is determined, defined sequence oligonucleotides that include a part of these genes may be synthesized. A 22-base DNA hybridization probe was produced for each of 2 heatstable E. coli enterotoxin (ST) genes: STH, from strains originally isolated from humans; and STP, from strains first found in pigs. For this study, 32P end-labeled DNA probes, sonicated calf thymus DNA, and 3 known and 20 unknown (10 ST-positive and 10 ST-negative) strains were sent to each of 23 collaborators. Cultures were spotted onto an agar-based medium and grown into colonies, which were transferred by blotting to cellulose filters, lysed by alkali and steam, and used for DNA colony hybridization with the ST DNA probes. Strains containing an ST gene were recognized as dark spots on an autoradiogram. Of the 460 samples analyzed, 440 (95.7%) were correctly classified by the collaborators. The method has been adopted official first action.

Stabilities of uncomplemented and complemented M15 β-galactosidase (Escherichia coli) and the relationship to α-complementation

1999 ◽  
Vol 77 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Clare N Gallagher ◽  
Reuben E Huber
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Thermal Inactivation ◽  
Quaternary Structure ◽  
Mutant Form ◽  
Narrow Temperature Range ◽  
Preferential Binding ◽  
High Concentrations ◽  
The Relationship ◽  
Tetrameric Form

M15 β-galactosidase (Escherichia coli) is a mutant form of β-galactosidase having residues 11-41 deleted. It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues. The activities of uncomplemented and complemented M15 β-galactosidases decreased starting at 42°C-uncomplemented over a narrow temperature range, complemented over a broad range. This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures. The effects of added components on stability and α-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation. Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10× less Mg2+ was needed for complemented protein). α-Complementation increased somewhat at low Mg2+ but decreased at high Mg2+. These effects can be explained by differential Mg2+ binding to the native and susceptible forms. The enhancement of both stability and α-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins. Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 β-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 β-galactosidase. α-Complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and α-complementation at low 2-mercaptoethanol owing to interactions with native forms. The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol. In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small. This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation. Key words: β-galactosidase, α-complementation, stability.

scholarly journals Characterization of Borrelia burgdorferiBlyA and BlyB Proteins: a Prophage-Encoded Holin-Like System

2000 ◽  
Vol 182 (23) ◽  
pp. 6791-6797 ◽  
Author(s):  
Christopher J. Damman ◽  
Christian H. Eggers ◽  
D. Scott Samuels ◽  
Donald B. Oliver
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Activity ◽  
Soluble Protein ◽  
Structural Similarity ◽  
Structural Features ◽  
Accessory Protein ◽  
E Coli ◽  
Bacteriophage Particle ◽  
Membrane Interactive

ABSTRACT The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308–7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. AnEscherichia coli mutant containing the blyABlocus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction ofsheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage inB. burgdorferi dramatically stimulated blyABexpression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.

Association of Enterohemorrhagic Escherichia coliHemolysin with Serotypes of Shiga-Like-Toxin-ProducingEscherichia coli of Human and Bovine Origins

1998 ◽  
Vol 64 (11) ◽  
pp. 4134-4141 ◽  
Author(s):  
Carlton Gyles ◽  
Roger Johnson ◽  
Anli Gao ◽  
Kim Ziebell ◽  
Denis Pierard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Severe Disease ◽  
Sheep Erythrocyte ◽  
Pcr Amplification ◽  
E Coli ◽  
Hemolysin Gene ◽  
Group 3 ◽  
Group 2 ◽  
Bovine Feces ◽  
Group 1

ABSTRACT In this study we investigated whether the enterohemorrhagicEscherichia coli (EHEC) hemolysin gene ehxAcould be used as an indicator of pathogenicity in Shiga-like-toxin-producing Escherichia coli (SLTEC) isolates. The isolates in a collection of 770 SLTEC strains of human and bovine origins were assigned to group 1 (230 human and 138 bovine SLTEC isolates belonging to serotypes frequently implicated in human disease), group 2 (85 human and 183 bovine isolates belonging to serotypes less frequently implicated in disease), and group 3 (134 bovine isolates belonging to serotypes not implicated in disease). PCR amplification was used to examine all of the SLTEC isolates for the presence of ehxA and the virulence-associated geneseae, slt-I, and slt-II. The percentages of human isolates in groups 1 and 2 that were positive forehxA were 89 and 46%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive forehxA were 89, 51, and 52%, respectively. The percentages of human isolates in groups 1 and 2 that were positive foreae were 92 and 27%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for eaewere 78, 15, and 19%, respectively. The frequencies of bothehxA and eae were significantly higher for group 1 isolates than for group 2 isolates. The presence of the ehxA gene was associated with serotype, as was the presence of the eae gene. Some serotypes, such as O117:H4, lacked both eae and ehxA and have been associated with severe disease, but only infrequently. Theslt-I genes were more frequent in group 1 isolates than in group 2 isolates, and the slt-II genes were more frequent in group 2 isolates than in group 1 isolates. In a second experiment we determined the occurrence of the ehxA andslt genes in E. coli isolated from bovine feces. Fecal samples from 175 animals were streaked onto washed sheep erythrocyte agar plates. Eight E. coli-like colonies representing all of the morphological types were transferred to MacConkey agar. A total of 1,080 E. coli isolates were examined, and the ehxA gene was detected in 12 independent strains, only 3 of which were positive for slt. We concluded that the ehxA gene was less correlated with virulence than the eae gene was and that EHEC hemolysin alone has limited value for screening bovine feces for pathogenic SLTEC because of presence of the ehxA gene in bovine isolates that are not SLTEC.

The effect of fasting and diet on fecal shedding of Escherichia coli O157:H7 by cattle

2000 ◽  
Vol 80 (4) ◽  
pp. 741-744 ◽  
Author(s):  
S. J. Buchko ◽  
R. A. Holley ◽  
W. O. Olson ◽  
V. P. J. Gannon ◽  
D. M. Veira
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Escherichia Coli O157 ◽  
Fecal Shedding ◽  
E Coli ◽  
Feed Withdrawal ◽  
Alfalfa Silage ◽  
Key Words Escherichia Coli

Cattle naturally infected with Escherichia coli O157:H7 were used to assess the effects of diet and feed withdrawal on the fecal shedding of E. coli O157:H7. Animals were fed an 80% concentrate diet (80% barley and 20% alfalfa silage), fasted for 48 h, fed a 100% forage diet (alfalfa silage), fasted for 48 h, and subsequently re-fed 100% forage (alfalfa silage). There were no differences in the numbers of animals positive for the shedding of E. coli O157:H7 when fed an 80% barley diet or an all-forage diet (P > 0.05) or during the fasting periods following each diet (P > 0.05). Upon re-feeding an all-forage diet following a 48-h fast, animals positive for E. coli O157:H7 shedding increased (P < 0.05), with 42.5% of the animals shedding the pathogen after 5 d. Re-feeding 100% forage following fasting appeared to have increased the number of animals shedding E. coli O157:H7 in their feces, which may have been influenced by diet in addition to fasting. Key words: Escherichia coli O157:H7, fasting, diet, cattle, fecal shedding

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