INFLUENCE OF IRON-LIMITED CONTINUOUS-CULTURE ON PHYSIOLOGY AND VIRULENCE OF LEGIONELLA-PNEUMOPHILA

A virulent strain of Legionella pneumophila serogroup 1, subgroup Pont iac, was grown in continuous culture at a constant growth rate under i ron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically define d medium. Residual contaminating iron, 0.45 mu M, was sufficient to su pport iron-limited growth. Typical iron-replete cultures metabolized 3 .3 mu M iron. Serine provided the principal source of carbon and energ y for both cultures, although iron-replete cultures also depleted a nu mber of other amino acids. There was a 40% decrease in culture biomass under iron restricted conditions. iron limitation did not significant ly affect carbohydrate metabolism, with the molar growth yield for car bon (Y-carbon) comparable for both cultures, However, under iron-limit ed conditions a sixfold increase in Y-iron correlated with a significa nt decrease in the iron content of the biomass, as the culture utilize d the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to i ron-deficient conditions. In addition to the morphological and physiol ogical changes, iron limitation had a critical effect on culture virul ence. The virulence of this strain was significantly (P < 0.05) reduce d when the culture was subjected to iron-limited conditions. This phen omenon was reversible, with a significant increase in culture virulenc e upon reversion to iron-replete conditions. When compared in an in vi tro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results furt her support the role of environmental parameters in regulating the vir ulence of L. pneumophila.