Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase

Abstract

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.

Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa

“…1B). Earlier characterization of algA, which codes for PMI, showe4 that the transcription of this gene was very low in both mucoid and nonmucoid strains, below the detection limits of RNA-DNA dot blot hybridization (20). However, using the insert of recombinant plasmid pAD2 as a probe (Fig.…”

“…Steps E, F, G, and I are putative enzymatic activities involved in the steps of polymerization, epimerization, and export through the cell membrane. Closed triangles denote enzymatic steps conclusively determined here or elsewhere (9,20). (B) Chromosomal regions with alg mutations and genes.…”

“…(B) Chromosomal regions with alg mutations and genes. Genes coding for PMI, algA (20), and GMD, algD, are located at the extreme ends of the alginate gene cluster (10) mapped at 45 min of the P. aeruginosa PAO chromosomal map. The position of these two genes corresponds to the locations of alg43 and alg-5 mutations, respectively (10).…”

“…In all samples protein concentration was determined by the method of Bradford (3). Assay of GMD in cell-free P. aeruginosa and E. coli extracts prepared as described before (20) was performed according to published procedures (38). GDPmannose-dependent reduction of NAD+ was monitored by following A340.…”

“…Our laboratory has demonstrated that, with the exception of one mutation (alg-22) mapping near the argH gene at 19 min of the P. aeruginosa chromosome (8), most of the mutations affecting alginate production are located near the argF gene at 45 min, forming a cluster that can be subdivided into at least six comnplementation groups (10). One gene, algA, was functionally identified in this cluster by intergeneric complementation (9) and direct assaying of the overproduced gene product (20) as coding for phosphomannose isomerase * Corresponding author.…”