Transcript Lecture-Mic 623-Plasmids-Corynebacterium - Home

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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.
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 0.2-150 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
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=305
227
Plasmid-vector for genetic engineering
http:// ct330/nf C.diphtheriae.jpegwww.bact.wisc.edu/Ba
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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-33615
Bielefeld, Germany.
[email protected]
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
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&
dopt=Abstract&list_uids=15336488&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 erythromycinresistant 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/µ g of phage
DNA, and transformation frequencies were in
the range of 0.2-150 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.
http://www.pnas.org/cgi/content/abstract/84/14/4964
Corynebacterium diphtheriae genes required
for acquisition of iron from haemin and
haemoglobin are homologous to ABC
haemin transporters
E. Susan Drazek1, Craig A. Hammack1 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.
http://www.blackwell-synergy.com/doi/abs/10.1046/j.13652958.2000.01818.x