Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 1'5S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglI-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability, With the algD-xyE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor.

Pulmonary infection by mucoid, alginate producing, Pseudomonas aeruginosa is a major complication in patients suffering from cystic fibrosis (CF). To analyze the mechanisms leading to the emergence of mucoid P. aeruginosa in CF lungs, control of the algD gene coding for GDPmannose dehydrogenase was studied. Transcriptional activation of algD was shown to be necessary for alginate production. Sequencing of algD and its promoter revealed multiple direct repeats upstream of the transcription start and throughout the promoter region. Using the algD-xy1E transcriptional fusion the algD promoter was demonstrated to be under positive control by the algR gene. This gene has previously been shown to undergo antibiotic promoted chromosomal amplification resulting in the emergence of the mucoid phenotype. These findings provide a basis for better understanding the control of mucoidy in P. aeruginosa.

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Phosphomannose isomerase activity was undetectable in extracts of mucoid (alginate-producing) Pseudomonas aeruginosa. When a P. aeruginosa gene previously shown to complement an alginate-negative mutant was overexpressed under the control of the tac promoter in the broad-host-range controlled-expression vector pMMB22, phosphomannose isomerase activity could be measured in extracts of P. aeruginosa and in a manA (phosphomannose isomerase-negative) mutant of Escherichia coli. P. aeruginosa extracts containing induced levels of enzyme were shown to interconvert fructose 6-phosphate and mannose 6-phosphate. A 56,000-dalton polypeptide was visualized on sodium dodecyl sulfate-polyacrylamide gels after induction in both hosts. When RNA-DNA dot-blot hybridization analysis was used, transcription of algA, the gene coding for P. aeruginosa phosphomannose isomerase, was not measurable from the chromosomes of either mucoid or nonmucoid P. aeruginosa. However, a high level of algA transcription was detected after expression of algA under tac promoter control in pMMB22.

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