Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.

Abstract:

:To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.

journal_name

Infect Immun

journal_title

Infection and immunity

authors

Kolodrubetz D,Phillips LH,Ezzo PJ,Kraig E

doi

10.1128/IAI.63.7.2780-2784.1995

subject

Has Abstract

pub_date

1995-07-01 00:00:00

pages

2780-4

issue

7

eissn

0019-9567

issn

1098-5522

journal_volume

63

pub_type

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