Transposon Tn5 mutagenesis in Pseudomonas aeruginosa: a study of Beta-lactamase expression

The object was to identify genes regulating expression of the chromosomally-encoded, inducible class C B-lactamase gene of P. aeruginosa. Transposon mutagenesis was considered an effective method for the identification and subsequent cloning of such regulatory genes. To facilitate identification of Tn5 insertions in P. aeruginosa a transposon Tn5-derivative, Tn5::gen, encoding gentamicin-resistance was constructed. Transposon Tn5::gen was transposed into a temperature-sensitive broad host-range plasmid, pMMB19. The resulting plasmid, pRTG1, was able to deliver Tn5::gen into the chromosome of E. coli. Attempts to introduce pRTG1 into P. aeruginosa were generally unsuccessful and recombination events were observed in most transconjugants tested. Spot-mating conjugation was capable of introducing pRTG1 into P. aeruginosa albeit at very low frequencies. Analysis of the resulting transconjugant plasmids showed pRTG1 was probably subject to a stepwise series of recombination events, sometimes resulting in total loss of plasmid within P. aeruginosa. One derivative, pRTGla, retained temperature-sensitivity and was used to deliver Tn5::gen into the P. aeruginosa chromosome. After growth at the non-permissive temperature, B-lactamase hyperproducing mutants of P. aeruginosa were isolated. Transposon Tn5::gen was located in the same chromosomal position in these mutants. Kinetic analysis of B-lactamase from these hyperproducing mutants showed them to have different basal B-lactamase activities and it seemed unlikely that Tn5::gen had a direct effect on B-lactamase synthesis. However, the basal B-lactamase activities of these mutants indicated that there were possibly six different basal activities to which P. aeruginosa can mutate.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• Allaway, David

Contributors dc:contributor

• Woodward, Martin J.
• Nichols, Wright W.
• King, Linda A.

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/xnhv-fg91

OAI identifier oai:identifier

tle:4da93d2e-91ec-4e8f-9954-04dcc262db8b:d6bd9758-527a-46cd-bfe2-c433766e8fca:1