Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H 2 O 2 , high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.Legionella dumoffii was first isolated from cooling-tower water in 1979 (18) and later from a postmortem lung specimen in the same year (40) as an atypical Legionella-like organism. It was later classified by Brenner (11) as a new species, L. dumoffii. Legionella species are gram-negative, facultative intracellular parasites of freshwater amoebae in nature and are capable of growing within alveolar macrophages and epithelial cells after being accidentally transmitted to humans (22). The most common human pathogen in the genus Legionella is L. pneumophila, the causative agent of Legionnaires' disease (71). Humans contract the disease from contaminated environmental sources, primarily by aspiration of aerosolized water sources (22). After internalization by alveolar macrophages, L. pneumophila-containing phagosomes do not acidify (34) or fuse with lysosomes (33). Instead, the mitochondria, smooth vesicles, and rough endoplasmic reticula (RER) near these L. pneumophila-containing vacuoles are recruited, and L. pneumophila begins to multiply in this unique niche (32). This altered endocytic pathway is considered to be controlled by the Dot/Icm type IV protein secretion system (5,17,48,55,56,74). The dot/icm genes are essential for the intracellular growth of L. pneumophila (5,51,60). T...
