Localization of the control region for expression of exotoxin A in Pseudomonas aeruginosa.

Abstract:

:The 2,760-base-pair (bp) PstI-EcoRI segment of the chromosome of Pseudomonas aeruginosa PA103 which carries the exotoxin A structural gene was expressed from an internal promoter when cloned in a pUC9 derivative and transformed into a nontoxigenic mutant of P. aeruginosa PAO1. The unique terminal EcoRI site was deleted, and a new EcoRI site was substituted for a PvuI site located 107 bp 5' to the transcription initiation site. Following EcoRI cleavage, Bal31 deletions were generated from this site, and an EcoRI linker sequence, GGAATTCC, was inserted in place of the deleted DNA. Mutants with deletions located 73 bp or more upstream of the transcription initiation site retained normal expression, whereas in mutants with deletions extending into the region 69 bp or less upstream of this site, exotoxin synthesis was greatly reduced. From a KpnI site located 473 bp 3' to the transcription initiation site, a similar series of Bal31 deletion mutants were generated in which the inserted EcoRI linker sequence was located within the same 72-bp region. Pairs of mutants from the two deletion series were identified in which the EcoRI linker was located at the same sequence, and these mutant pairs were ligated to derive a series of constructs in which the EcoRI linker sequence GGAATTCC was substituted for an 8-bp sequence within the 72-bp region. Some of these linker-substituted mutants showed greatly reduced exotoxin A synthesis. The results are consistent with a binding site for a positive activator contiguous with the binding site for an RNA polymerase.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Tsaur ML,Clowes RC

doi

10.1128/jb.171.5.2599-2604.1989

subject

Has Abstract

pub_date

1989-05-01 00:00:00

pages

2599-604

issue

5

eissn

0021-9193

issn

1098-5530

journal_volume

171

pub_type

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