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 .3 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 103 plaque-forming units/pg of phage DNA, and transformation frequencies were in the range of 0.2-150 colony-forming units/
The relationship of plasmid pNG2, isolated from an erythromycin-resistant strain of Corynebactenium diphtheriae, to plasmids isolated from skin coryneforms was examined. The extent of homology between plasmids from erythromycin-resistant and -susceptible skin coryneforms and pNG2 varied, but in aggregate homology was observed with all six BstEII fragments of pNG2. The data support the hypothesis that pNG2 originated in skin coryneforms. Intact plasmid pNG2 and some of its restriction fragments were cloned into Escherichia coli JM109. The erythromycin resistance phenotype was expressed in clones carrying intact pNG2 as well as in some of its fragments and appeared to depend on a C. diphtheriae promoter for expression. A 2.5-megadalton EcoRI fragment, the smallest expressing resistance, contained the 1.2-megadalton region of pNG2 which is deleted when the erythromycin-resistant strain of C. diphtheriae reverts spontaneously to the susceptible state.Schiller et al. (16) isolated the first and thus far only Corynebacterium diphtheriae plasmid from clinical strains that were resistant to erythromycin (Emr). The plasmid, pNG2, has a molecular mass of 9.6 megadaltons (MDa), and the data suggest that the plasmid gene(s) mediating resistance is on a 1.2-MDa segment that is consistently lost when Emr strains revert spontaneously to the erythromycinsusceptible (Ems) state. The susceptible revertants carry the 8.4-MDa plasmid pNG3 in place of
Subcloning and protoplast transformation studies identified a 2.6 kb fragment of Corynebacterium diphtheriae plasmid pNG2 which contains an origin of replication (oriR). Molecular combination of the 2.6 kb oriR cartridge with Escherichia coli plasmid pUC18CmR enabled the E. coli cloning vector to replicate within several species of Corynebacterium host cells. A 2.6 kb plasmid formed from the oriR cartridge alone is capable of replicating in E. coli. This suggests that a single origin could be used in vectors shuttling between Corynebacterium spp. and E. coli.
Plant tissues often contain I8-glucosides that can be enzymatically hydrolyzed to produce toxic aglycones. It has been suggested that the low i-glucosidase activity found in Erwinia amylovora contributes to bacterial virulence by allowing the bacteria to infect plants that contain 13-glucosides without inducing the formation of toxic aglycones. To test this suggestion, we created strains of E. amylovora which had high 1-glucosidase activities and studied the ability of these strains to cause fire blight disease in pears (Pyrus communis). We isolated spontaneous mutants that were able to utilize ,3-glucosides as the sole carbon source and showed that one class had about 10 times as much ,3-glucosidase activity as the wild-type strain. In addition, we constructed several plasmids that carry the Escherichia coli bgl operon under the control of a transposon TnS promoter that is expressed in E. amylovora. These plasmids were introduced in E. amylovora by transformation. Pathogenesis studies in immature Bartlett pear fruits, etiolated sprouts, and young shoots showed that a 100-fold increase in 3-glucosidase activity does not interfere with normal development of fire blight disease in these model systems. LB medium supplemented with 1 mM MgCl2-0.5 mM CaCl2, washed first with 100 mM MgCl2 and then with 50 mM 677
A 9.6 Mda Corynebacterium diphtheriae plasmid, pNG2, mediates inducible resistance to erythromycin and other macrolide‐lincosamide‐streptogramin B (MLS) antibiotics. Spontaneous deletion of a 1.2 Mda fragment of pNG2 leaves the derivative plasmid incapable of conferring MLS‐resistance on its host. The pNG2 region containing this fragment was sequenced as were the regions flanking it. The fragment contained 1606 bp, was flanked by almost perfect 23 bp inverted repeats, and appeared to be excised precisely. It contained an open reading frame encoding a leader protein plus a protein which was similar in its amino acid sequence to the rRNA methylases produced by other erythromycin‐resistant, Gram‐positive bacteria. The C. diphtheriae gene was named ermCd.
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