Cytochrome c 4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c 1 and c 5 promote intracellular infection

Abstract

A panel of cytochromecmaturation (ccm) mutants ofLegionella pneumophiladisplayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and aLegionella-specific bioassay. These data, coupled with the finding thatccmtranscripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression byL. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individualc-type cytochromes. Whereas ubiquinol-cytochromecreductase (petC) mutants specifically lacking cytochromec1andcycBmutants lacking cytochromec5had normal siderophore expression,cyc4mutants defective for cytochromec4completely lacked legiobactin. These data, along with the expression pattern ofcyc4mRNA, indicate that cytochromec4in particular promotes siderophore expression. In intracellular infection assays,petCmutants andcycBmutants, but notcyc4mutants, had a reduced ability to infect both amoebae and macrophage hosts. Likeccmmutants, thecycBmutants were completely unable to grow in amoebae, highlighting a major role for cytochromec5in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of ac-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance ofc-type cytochromes in intracellular infection events.