FUMARASE-C ACTIVITY IS ELEVATED IN RESPONSE TO IRON DEPRIVATION AND IN MUCOID, ALGINATE-PRODUCING PSEUDOMONAS-AERUGINOSA - CLONING AND CHARACTERIZATION OF FUMC AND PURIFICATION OF NATIVE FUMC

We report the discovery of fumC, encoding a fumarase, upstream of the sodA gene, encoding manganese superoxide dismutase, in Pseudomonas aer uginosa. The fumC open reading frame, which terminates 485 bp upstream of sodA, contains 1,374 bp that encode 458 amino acids. A second 444- bp open reading frame located between fumC and sodA, called orfX, show ed no homology with any genes or proteins in database searches. A fuma rase activity stain revealed that P. aeruginosa possesses at least two and possibly three fumarases. Total fumarase activity was at least si milar to 1.6-fold greater in mucoid, alginate-producing bacteria than in nonmucoid bacteria and decreased 84 to 95% during the first 5 h of aerobic growth, followed by a rapid rise to maximum activity in statio nary phase. Bacteria exposed to the iron chelator 2,2'-dipyridyl, hut not ferric chloride, demonstrated an increase in fumarase activity. Mu coid bacteria produced approximately twofold-higher levels of the side rophores pyoverdin and pyochelin than nonmucoid bacteria, Northern blo t analysis revealed a transcript that included fumC, orfX, and sodA, t he amount of which was increased in response to iron deprivation. A P. aeruginosa fumC mutant produced only similar to 40% the alginate of w ild-type bacteria. Interestingly, a sodA mutant possessed an alginate- stable phenotype, a trait that is typically unstable in vitro. These d ata suggest that mucoid bacteria either are in an iron-starved state r elative to nonmucoid bacteria or simply require more iron for the proc ess of alginate biosynthesis. In addition, the iran-regulated, tricarb oxylic acid cycle enzyme fumarase C is essential for optimal alginate production by P. aeruginosa.