We exploited Pseudomonas cepacia's inability to utilize lactose to isolate strains in which transposable gene-activating elements from this bacterium activated the expression of the lac genes of Tn951 and permitted growth on lactose. Tn951 was introduced into P. cepacia on the broad-host-range plasmid pGC91.14. The lac genes of this tranposon were not expressed in P. cepacia. However, Lac$\sp{+}$ variants in which $\beta$-galactosidase was formed constitutively at high levels were isolated at a frequency of ca. 10$\sp{-6}$. RNA-DNA hybridization analysis indicated that the elevated levels of $\beta$-galactosidase in the Lac$\sp{+}$ variants were due to a marked increase in the levels of lac-specific mRNA. The plasmids of the three different Lac$\sp{+}$ strains were transferred by conjugation to Pseudomonas aeruginosa and a lac strain of Escherichia coli and the constitutive expressions of lacZ gene was observed in these strains. The plasmids were subjected to restriction enzyme analysis and found to contain additional DNA not present in pGC91.14. The insertions were due to the transposition of IS elements IS406, IS407 and IS415 from the P. cepacia chromosome to sites upstream of the lac structural genes of Tn951. Nucleotide sequence analysis of IS407 (1236 bp) confirmed the presence of features characteristics of IS-elements: terminal inverted repeat sequences and the generation of nucleotide sequence duplications at the target site. Sequence analysis of the lac promoter-operator region of pTGL67 (pGC91.14::IS407) placed the insertion site of IS407 within lacO. The insertion of IS407 separated $lacP\sb{\rm Tn951}$ from lacZ by ca. 1.3 kb. The nucleotide sequence of IS407 revealed the presence of an outwardly directed promoter proximate to the terminal inverted repeat adjacent to lacZ. Primer extension analysis of lacZ-specific mRNA isolated from E. coli determined that this promoter was operating in vivo and responsible for the constitutive expression of lacZ in this bacterium.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-7718 |
| Date | 01 January 1990 |
| Creators | Wood, Mark Stephen |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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