Pattern of changes in the activity of enzymes of GDP-D-mannuronic acid synthesis and in the level of transcription of algA, algC and algD genes accompanying the loss and emergence of mucoidy in Pseudomonas aeruginosa

The low activity levels of the four GDP-D-mannuronic acid-forming enzymes, even in highly alginate-producing strains of Pseudomonas aeruginosa, have m ade it difficult to compare enzyme activities accompanying the loss/acquisi tion of mucoidy. Using optimized conditions, we compared the specific activ ity of these enzymes in three different mucoid P. aeruginosa cystic fibrosi s isolates, in their nonmucoid spontaneous variants, and in mucoid variants that emerged during extended incubation of these nonmucoid forms in acetam ide broth. A correlation was established between the promptness of emergenc e of the mucoid forms and the differing sensitivity to nutrient-limitation- induced death of the nonmucoid compared with the isogenic mucoid population . Consistent with the undetectable levels of algD mRNA in nonmucoid forms a nd with the concept that the step catalyzed by the algD-encoded GDP-mannose dehydrogenase (GMD) is a key step in control of the alginate pathway, GMD activity was undetectable or showed negligible values in nonmucoid variants and correlated with alginate production. However, phosphomannose isomerase (PMI), phosphomannomutase (PMM), and GDP-mannose pyrophosphorylase (GMP) a ctivities in the nonmucoid forms were only slightly (40-70%) below the valu es in the mucoid forms. Nevertheless, no transcripts homologous to algA (en coding a bifunctional enzyme that possesses both PMI and GMP activities) we re detected in the nonmucoid form, and the levels of algC (encoding PMM) tr anscripts, although detectable in the nonmucoid variants, were, in general, much higher in the mucoid forms. These apparently intriguing observations were cleared up by the identification of two algA functional homologues in P. aeruginosa, recently reported by others, and by the identification of on e algC homologue, in contig225 of the PAO1 genome sequence, defining a poly peptide with a deduced amino acid sequence that showed significant homology with that of enzymes of the phosphohexomutase family found in databases. R esults are also consistent with the requirement of PMI, GMP and PMM activit ies for the supply of GDP-D-mannose to (at least) A-band Lipopolysaccharide synthesis, while GMD channels this precursor into the alginate pathway. (C ) Elsevier, Paris.