Cysteine Metabolism in Legionella pneumophila : Characterization of an l -Cystine-Utilizing Mutant

Abstract

ABSTRACT Growth of Legionella pneumophila on buffered charcoal-yeast extract (BCYE) medium is dependent on l -cysteine (but not l -cystine), which is added in excess over what is required for nutrition. We investigated the biochemical and genetic bases for this unusual requirement and determined that much of the l -cysteine in BCYE medium is rapidly oxidized to l -cystine and is unavailable to the bacteria. Analysis of cysteine consumption during bacterial growth indicated that of the 11% consumed, 3.85% (∼0.1 mM) was incorporated into biomass. The activities of two key cysteine biosynthetic enzymes (serine acetyltransferase and cysteine synthase) were not detected in cell extracts of L. pneumophila , and the respective genes were not present in the genome sequences, confirming cysteine auxotrophy. Kinetic studies identified two energy-dependent cysteine transporters, one with high affinity (apparent K m , 3.29 μM) and the other with low affinity (apparent K m , 93 μM), each of which was inhibited by the uncoupling agent carbonyl cyanide m -chlorophenylhydrazone. Cystine was not transported by L. pneumophila ; however, a mutant strain capable of growth on l -cystine (CYS1 mutant) transported l -cystine with similar kinetics ( K m , 4.4 μM and 90 μM). Based on the bipartite kinetics, requirement for proton motive force, and inhibitor studies, we suggest that a high-affinity periplasmic binding protein and a major facilitator/symporter (low affinity) mediate uptake. The latter most likely is functional at high cysteine concentrations and most likely displays altered substrate specificity in the CYS-1 mutant. Our studies provide biochemical evidence to support a general view that L. pneumophila is restricted to an intracellular lifestyle in natural environments by an inability to utilize cystine, which most likely ensures that the dormant cyst-like transmissible forms do not germinate outside suitable protozoan hosts.