UTILIZATION OF A MINI-DLAC TRANSPOSABLE ELEMENT TO CREATE AN ALPHA-COMPLEMENTATION AND REGULATED EXPRESSION SYSTEM FOR CLONING IN PSEUDOMONAS-AERUGINOSA
Rr. Karkhoffschweizer et Hp. Schweizer, UTILIZATION OF A MINI-DLAC TRANSPOSABLE ELEMENT TO CREATE AN ALPHA-COMPLEMENTATION AND REGULATED EXPRESSION SYSTEM FOR CLONING IN PSEUDOMONAS-AERUGINOSA, Gene, 140(1), 1994, pp. 7-15
A lac-based alpha-complementation and expression system was developed
for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophag
e D3ll2-based mini-Dlac transposable element, containing the lacI(q)-r
egulated lazZ Delta M15 gene next to a selectable marker, was construc
ted. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, an
d derivatives containing randomly inserted chromosomal copies of the m
ini-Dine element were obtained. Transformation of the PAO1::mini-Dlac
transductants with the broad-host-range vector, pUCP19, led to the for
mation of blue colonies on indicator medium in the presence of inducer
. In contrast, transformants harboring the pUCP19 derivative pCDO, con
taining the catechol-2,3-dioxygenase (C230)-encoding xy/E gene under I
nc promoter control, were white on the same medium. Expression of xy/E
was tightly controlled by single-copy mini-Dine-encoded inc repressor
and in induced cultures was increased more than 100-fold over that ob
served in uninduced cultures. The usefulness of the system for molecul
ar cloning in P. aeruginosa was demonstrated by ligating size-fraction
ated PAO1 chromosomal fragments into pUCP19, followed by transformatio
n of the newly isolated PAO1::mini-Dlac host. All randomly chosen whit
e colonies contained recombinant plasmids, with inserts of the correct
size range, while blue colonies contained pUCP19 alone. The functiona
lity of the system was also shown in another frequently studied strain
, PA103.