The Cytochrome c Maturation Locus of Legionella pneumophila Promotes Iron Assimilation and Intracellular Infection and Contains a Strain-Specific Insertion Sequence Element

Abstract

Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.

lbtAandlbtBAre Required for Production of theLegionella pneumophilaSiderophore Legiobactin

“…Although FrgA is homologous with hydroxamate synthetases and frgA promotes growth within the iron-limited intracellular niche in macrophages (35), frgA mutants proved not to be defective for CAS reactivity when grown in low-iron CDM (39). We also observed that the cytochrome c maturation operon, which promotes L. pneumophila growth in low-iron media as well as siderophore expression in several other types of bacteria, is not required for legiobactin production (47,82). In another recent study, the feoB ferrous iron transporter showed no role in the generation of L. pneumophila CAS reactivity (63).…”

Indole-3-Acetic Acid Biosynthesis Is Deficient in Gluconacetobacter diazotrophicus Strains with Mutations in Cytochrome c Biogenesis Genes

“…Additionally, in some prokaryotes, a defect in the biogenesis of cytochrome c leads to a dramatic increase in synthesis and excretion of heme biosynthetic intermediates (10,21). Other reported phenotypes of cytochrome c mutants are loss of copper resistance (51) and pyoverdine production (19) in Pseudomonas fluorescens, defects in nitrogen fixation in B. japonicum and R. phaseoli (36,43), and reduced intracellular infection in Legionella pneumophila (50), as well as defects in high-affinity iron acquisition in Rhizobium leguminosarum and P. fluorescens (9,52). However, there are no reports describing the relationship between IAA production and cytochrome c in the literature.…”

Heme Concentration Dependence and Metalloporphyrin Inhibition of the System I and II CytochromecAssembly Pathways

“…Because heme biosynthesis depends on central metabolism (e.g., glutamate or succinyl coenzyme A and glycine), a limiting factor for heme biosynthesis is often the free iron levels (27,42,56). Based on the presence of the "heme reservoir" CcmE (24) and the fivefold-higher affinity for heme, we speculate that organisms in which heme synthesis is limited, such as organisms faced with low-iron environments, would have an advantage using system I.…”