A panel of
cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of
siderophore (legiobactin) expression, as measured by both the
chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes
siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type
cytochromes. Whereas
ubiquinol-cytochrome c reductase (petC) mutants specifically lacking
cytochrome c(1) and cycB mutants lacking
cytochrome c(5) had normal
siderophore expression, cyc4 mutants defective for
cytochrome c(4) completely lacked legiobactin. These data, along with the expression pattern of cyc4
mRNA, indicate that
cytochrome c(4) in particular promotes
siderophore expression. In intracellular
infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for
cytochrome c(5) in intracellular
infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type
cytochrome in expression of a biologically active
siderophore and the first insight into the relative importance of c-type
cytochromes in intracellular
infection events.