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Mol Gen Genet. 1991 Aug;228(1-2):177-82.

Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA.

Author information

1
Institut für Genetik und Mikrobiologie, Universität Würzburg, Federal Republic of Germany.

Abstract

A plasmid integration technique was developed for insertional inactivation of chromosomal Listeria monocytogenes genes. A Listeria-Escherichia coli shuttle vector (pLSV1) was constructed which carried the temperature-sensitive gram-positive replication origin from plasmid pTV32(Ts). An internal fragment of the listeriolysin gene (lisA) was cloned into pLSV1 to create pLSV2. In L. monocytogenes pLSV2 transformants, plasmid pLSV2 integrated into the L. monocytogenes chromosome at a frequency of 2 x 10(-3) via lisA homology and these cells could be selected at 42 degrees C using a plasmid-encoded erythromycin resistance. Plasmid integration resulted in disruption of the lisA gene, production of a truncated, immunologically cross-reactive listeriolysin protein and loss of the hemolytic phenotype. An improved Listeria protoplast transformation method is also described which facilitates genetic manipulation of Listeria species.

PMID:
1909419
DOI:
10.1007/bf00282463
[Indexed for MEDLINE]

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