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J. Biol. Chem., Vol. 269, Issue 31, 19787-19795, Aug, 1994
UA Ochsner, A Fiechter and J Reiser
Transposon Tn5-GM-induced mutant strains of Pseudomonas aeruginosa which
are unable to produce rhamnolipid biosurfactants and lack
rhamnosyltransferase activity have been isolated. The DNA regions flanking
the transposon were cloned and used as specific probes for the isolation of
the corresponding wild-type genes from a P. aeruginosa wild-type cosmid
gene library. Single cosmid clones capable of restoring rhamnolipid
synthesis in the mutant strains were isolated and further subcloned and
sequenced, resulting in the identification of two genes (rhlAB) which are
organized as an operon upstream of the previously identified rhlR
regulatory gene. The RhlA protein (32.5 kDa) harbors a putative signal
sequence, suggesting that this protein is located in the periplasm, while
the RhlB protein (47 kDa) contains at least two putative membrane-spanning
domains. The expression of the rhlAB genes was found to be enhanced 20-fold
during the stationary phase of growth under conditions of nitrogen
limitation, as measured by using rhlA::lacZ fusions. Moreover, the
transcriptional activation of the rhlAB genes appears to depend on a
functional RhlR regulatory protein. The sequence upstream of the rhlA
promoter contains two inverted repeats which define putative binding sites
for the RhlR regulator. The controlled expression of the rhlAB genes in
Escherichia coli led to the formation of active rhamnosyltransferase. This
provides direct evidence for the fact that the rhamnosyltransferase
encoding genes have been identified.
Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis
Institute for Biotechnology, Swiss Federal Institute of Technology, ETH- Honggerberg, Zurich.
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