Ce. Chitnis et De. Ohman, GENETIC-ANALYSIS OF THE ALGINATE BIOSYNTHETIC GENE-CLUSTER OF PSEUDOMONAS-AERUGINOSA SHOWS EVIDENCE OF AN OPERONIC STRUCTURE, Molecular microbiology, 8(3), 1993, pp. 583-590
Strains of Pseudomonas aeruginosa causing pulmonary infections in cyst
ic fibrosis patients have an unusual mucoid phenotype because of produ
ction of the capsule-like exopolysaccharide, alginate. Transcriptional
activation of algD, the first gene of a large alginate biosynthetic g
ene cluster, is associated with conversion to the alginate-producing (
Alg+) phenotype. In this study, we examined the regulation of alginate
genes immediately downstream of algD. Mutants of the Alg+ strain FRD1
were constructed by gene replacement with defined Tn501 (8.2 kb) inse
rtions in the alginate biosynthetic gene cluster, resulting in an Alg-
phenotype. The Alg+ phenotype of these mutants was restored by integr
ation of narrow-host-range plasmids containing DNA fragments from P. a
eruginosa that reconstructed a continuous alginate gene cluster. A bro
ad-host-range plasmid containing the entire alginate gene cluster exce
pt for the terminal gene, algA, was unable to complement an alG::Tn501
mutant unless algA was transcribed from a second plasmid. This indica
ted that any Tn501 insertion in the cluster was polar on downstream al
ginate genes. Northern blot hybridization experiments also showed that
a transposon insertion downstream of algD adversely affected algG and
algA transcription. These results provided evidence that the alginate
biosynthetic gene cluster has an operonic structure and is cotranscri
bed from the algD promoter.