أ.د. عالية عبد الباقي شعيب المملكة العربية السعودية جامعة الملك سعود كلية العلوم قسم البنات والأحياء الدقيقة Mic 623 Plasmids and Antibiotics in Pathogenic Bacteria

1- Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2. T M Serwold-Davis, N Groman, and M Rabin This article has been cited by other articles in PMC.cited by بِسْمِ اللهِ الرَّحْمنِ الرَّحِيمِ

Abstract The transfection and transformation of members of two species of pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, is described. Protoplasts were produced by treatment with lysozyme following growth in glycine, and a medium was defined on which a significant fraction of the osmotically sensitive cells were regenerated.

Transfections were carried out with DNA from corynephage 782, a member of the beta family of converting phages, and transformations were performed with DNA of plasmid pNG2, a 9500-kDa plasmid that was isolated from an erythromycin-resistant strain of C. diphtheriae and carries the resistance gene.

Strains of Corynebacterium glutamicum and Escherichia coli were also successfully transformed with pNG2 DNA.

Transfection frequencies were in the range of 3-8 X 10(3) plaque-forming units/micrograms of phage DNA, and transformation frequencies were in the range of colony-forming units/micrograms of plasmid DNA. Plasmid pNG2 replicated and was stably maintained in all transformants both in the presence or absence of erythromycin. Thus, it displayed the ability to replicate in strains of both Gram-positive and Gram-negative bacteria without the intervention of genetic engineering.

pNG2 DNA isolated from any of the transformed strains was able to transform all parental strains. The host range of pNG2 suggests its possible utility in or as a shuttle vector for the study and manipulation of genes from corynebacterial strains of animal origin. Full text 227

Plasmid-vector for genetic engineering

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Comparative genomics identified two conserved DNA modules in a corynebacterial plasmid family present in clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium. Tauch A, Bischoff N, Puhler A, Kalinowski J. Institut fur Genomforschung, Universitat Bielefeld, Universitatsstrasse 25, D Bielefeld, Germany. Tauch ABischoff NPuhler AKalinowski J

Investigation of 62 clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium revealed that 17 possessed plasmids ranging in size from 7.6 to 14.9 kb. The plasmids formed four groups on DNA restriction analysis.

The complete nucleotide sequence of a representative from each group (pK43, pK64, pCJ84, and pB85766) was subsequently determined.

Additionally, two plasmids (pCo455 and pCo420) were shown to be derivatives of pK43 and pK64 carrying insertion sequences of the IS3 family. Comparative genomics identified a conserved plasmid backbone consisting of two distinct DNA modules.

Conserved motifs in the parAB-repA module indicated that the sequenced plasmids from C. jeikeium are new members of the pNG2 family. Recombinant derivatives of pK43 were shown to replicate in the soil bacterium Corynebacterium glutamicum and in the human pathogen C.diphtheriae.

The second plasmid module most likely encodes a novel type of DNA invertase. The respective gene is flanked by highly conserved 112-bp inverted repeats. All plasmids are 'loaded' with a characteristic set of genes encoding products of unknown function

Plasmids indistinguishable from pK43 by DNA restriction analysis were identified in different C. jeikeium strains, which revealed 16S-23S rDNA spacer length polymorphisms and specific antibiotic susceptibility profiles, implying a wide dissemination of the plasmid in clinical isolates of C. jeikeium dopt=Abstract&list_uids= &query_hl=16&itool=pubmed_docsum

Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae Plasmid pNG2 Theresa M. Serwold-Davis, Neal Groman, and Myron Rabin The transfection and transformation of members of two species of pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, is described.

Protoplasts were produced by treatment with lysozyme following growth in glycine, and a medium was defined on which a significant fraction of the osmotically sensitive cells were regenerated.

Transfections were carried out with DNA from corynephage 782, a member of the ß family of converting phages, and transformations were performed with DNA of plasmid pNG2, a 9500-kDa plasmid that was isolated from an erythromycin- resistant strain of C. diphtheriae and carries the resistance gene.

Strains of Corynebacterium glutamicum and Escherichia coli were also successfully transformed with pNG2 DNA. Transfection frequencies were in the range of 3-8 x 10 3 plaque-forming units/µ g of phage DNA, and transformation frequencies were in the range of colony-forming units/µ g of plasmid DNA.

Plasmid pNG2 replicated and was stably maintained in all transformants both in the presence or absence of erythromycin.

Thus, it displayed the ability to replicate in strains of both Gram-positive and Gram- negative bacteria without the intervention of genetic engineering. pNG2 DNA isolated from any of the transformed strains was able to transform all parental strains.

The host range of pNG2 suggests its possible utility in or as a shuttle vector for the study and manipulation of genes from corynebacterial strains of animal origin.

Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters E. Susan Drazek 1, Craig A. Hammack 1 Sr, and Michael P. Schmitt* Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium.

C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants.

The complementing plasmids shared an ≈ 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon.

The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system.

The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae.

HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae.

This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram- positive bacterium x