Control of corynebacteriophage reproduction by heteroimmune repression.
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Corynebacteriophages beta and gamma are closely related but heteroimmune; hence, gamma reproduces in C7(beta). A series of gamma mutants, designated gamma-bin (beta-inhibited), has been isolated. They reproduce in only 2 to 14% of infected C7(beta) cells, and, as a result, plaque with an efficiency of 10(-4) to 10(-5) on this strain. The proportion of C7(beta) cells in which gamma-bin phage can replicate is increased to 30 to 80% when immunity is lifted by UV induction of C7(beta) or by heat induction of C7(beta-tsr3). The gamma-bin mutants carry out a normal vegetative or lysogenic cycle in strain C7 and thus do not appear to be defective in any essential phage function. Infection of C7(beta) by gamma-bin results in cell killing whether the infection is productive or nonproductive. The data support the hypothesis that inhibition of gamma-bin is due to the direct or indirect action of a beta prophage gene. The simplest hypothesis is that gamma-bin phages have sustained mutations in an operator site and that beta repressor now combines with the mutated operator to inhibit normal replication in a significant proportion of infected cells. (+info)
Use of molecular subtyping to document long-term persistence of Corynebacterium diphtheriae in South Dakota.
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Enhanced surveillance of patients with upper respiratory symptoms in a Northern Plains community revealed that approximately 4% of them were infected by toxigenic Corynebacterium diphtheriae of both mitis and gravis biotypes, showing that the organism is still circulating in the United States. Toxigenic C. diphtheriae was isolated from five members of four households. Four molecular subtyping methods-ribotyping, multilocus enzyme electrophoresis (MEE), random amplified polymorphic DNA (RAPD), and single-strand conformation polymorphism-were used to molecularly characterize these strains and compare them to 17 archival South Dakota strains dating back to 1973 through 1983 and to 5 isolates collected from residents of diverse regions of the United States. Ribotyping and RAPD clearly demonstrated the household transmission of isolates and provided precise information on the circulation of several distinct strains within three households. By MEE, most recent and archival South Dakota strains were identified as closely related and clustered within the newly identified ET (electrophoretic type) 215 complex. Furthermore, three recent South Dakota isolates and eight archival South Dakota isolates were indistinguishable by both ribotyping and RAPD. All of these molecular methods showed that recent South Dakota isolates and archival South Dakota isolates were more closely related to each other than to the C. diphtheriae strains isolated in other parts of the United States or worldwide. The data also supported the improbability of importation of C. diphtheriae into this area and rather strongly suggest the long-term persistence of the organism in this region. (+info)
Quantitative studies on competitive activities of skin bacteria growing on solid media.
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Earlier quantitative investigations of antagonism between skin bacteria were based on the use of liquid cultures, but a more realistic model has now been devised, based on the use of the surfaces of solid media. Pure or mixed inocula were spread evenly over suitable agar media in Petri dishes marked out with a standard grid. Growth curves were constructed from viable counts of the surface bacteria after they had been removed from excised squares of the agar media and dispersed. The method was highly reproducible, and competitive interactions were revealed more clearly than in studies with liquid media. An antibiotic-producing strain of Staphylococcus epidermidis (S6+) readily suppressed strains of Micrococcus, Corynebacterium and Streptococcus species. However, a Staphylococcus aureus strain which was less sensitive to the antibiotic effect of S6+ interacted in a complex manner, depending on the absolute and relative size of the S6+ inoculum. (+info)
Resurgent diphtheria--are we safe?
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Diphtheria, one of the major causes of morbidity and mortality in the past, seemed nearly eliminated from industrialized countries, thanks to improved hygienic conditions and large scale vaccinations. In 1990, a large epidemic started in Eastern Europe, mainly in Russia and Ukraine, with over 70,000 cases reported within a 5 year period. The main factors leading to the epidemic included low immunization coverage among infants and children, waning immunity to diphtheria among adults, and profound social changes in the former Soviet Union. The possibility of new virulence factors in the epidemic strain has not yet been ruled out. Even though immunity among adults is far from complete in Western Europe, the epidemic did not spread there. The main reason for this might be the good immune status of children and lack of social turbulence favouring the spread of infection. Several countries have also taken preventive measures, which may also have played a role in protection against the potential epidemic. (+info)
Emergence of related nontoxigenic Corynebacterium diphtheriae biotype mitis strains in Western Europe.
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We report on 17 isolates of Corynebacterium diphtheriae biotype mitis with related ribotypes from Switzerland, Germany, and France. Isolates came from skin and subcutaneous infections of injecting drug users, homeless persons, prisoners, and elderly orthopedic patients with joint prostheses or primary joint infections. Such isolates had only been observed in Switzerland. (+info)
Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.
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Hmu O, a heme degradation enzyme in the pathogen Corynebacterium diphtheriae, catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. A bacterial expression system using a synthetic gene coding for the 215-amino acid, full-length Hmu O has been constructed. Expressed at very high levels in Escherichia coli BL21, the enzyme binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converts hemin to biliverdin with alpha-hydroxyhemin and verdoheme as intermediates. The overall conversion rate to biliverdin is approximately 4-fold slower than that by rat heme oxygenase (HO) isoform 1. Reaction of the hemin-Hmu O complex with hydrogen peroxide yields a verdoheme species, the recovery of which is much less compared with rat HO-1. Reaction of the hemin complex with meta-chloroperbenzoic acid generates a ferryl oxo species. Thus, the catalytic intermediate species and the nature of the active form in the first oxygenation step of Hmu O appear to be similar to those of the mammalian HO. However, the considerably slow catalytic rate and low level of verdoheme recovery in the hydrogen peroxide reaction suggest that the active-site structure of Hmu O is different from that of its mammalian counterpart. (+info)
The heme complex of Hmu O, a bacterial heme degradation enzyme from Corynebacterium diphtheriae. Structure of the catalytic site.
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Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart. (+info)
Identification of a two-component signal transduction system from Corynebacterium diphtheriae that activates gene expression in response to the presence of heme and hemoglobin.
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Corynebacterium diphtheriae, the causative agent of diphtheria, utilizes various host compounds to acquire iron. The C. diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the hmuO gene in C. diphtheriae is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a C. diphtheriae chromosomal library were shown to activate transcription from the hmuO promoter in Escherichia coli. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the C. diphtheriae chromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (chrA) and its cognate sensor kinase (chrS), was shown to strongly activate transcription from the hmuO promoter in a heme-dependent manner in E. coli. A mutation in chrA (chrAD50N), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the hmuO promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism. (+info)