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Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella.

Legionella pneumophila, an intracellular parasite of macrophages and protozoa, requires iron for extra- and intracellular growth. In a new screen of a mutant library of L. pneumophila for strains defective for growth on agar media lacking supplemental iron, seven mutants were obtained. All of the mutants had a disruption in the cytochrome c maturation (ccm) locus; two had insertions in ccmB, two in ccmC, and three in ccmF. The ccm mutants were unable to multiply within macrophage-like cells (i.e., U937 and THP-1 cells) and Hartmannella vermiformis amoebae. A competition assay in A/J mice revealed that ccm mutants are severely defective for growth within the lung. Taken together, these data confirm that ccm and cytochrome c maturation proteins are required for L. pneumophila growth in low iron, intracellular infection, and virulence.

Unlike most beta-related phages isolated from Corynebacterium diphtheriae, phage 782 readily plaqued on strains of C. ulcerans and C. pseudotuberculosis. The extended host range of phage 782 was not, however, due to a greater ability of the phage to absorb to bacterial surfaces. Using chemical mutagenesis, a number of phage mutants were isolated which had diminished capacities to infect C. ulcerans, suggesting the existence of a locus (ehr) for extended host range. Pre-lysogenization of C. ulcerans strains with phage 782, but not its mutant form or other beta-related phages, rendered them susceptible to infection by previously excluded phages. An examination of recombinant between phages 782, pie, and beta localized ehr to a 7 kilobase region of DNA including attP. The data are compatible with the notion that ehr encodes an anti-restriction function.

To more effectively study the genetic basis of Legionnaires' disease, we characterized a system for mini-Tn10 mutagenesis in Legionella pneumophila. The mini-transposons were first electroporated into Legionella on counterselectable vectors expressing altered target site transposases. Then, by simultaneously selecting for the kanamycin-resistance gene within the transposon and counterselecting against the maintenance of the plasmid, we directly and readily isolated strains bearing single chromosomal insertions. Southern hybridization analysis further demonstrated that the insertions were randomly distributed throughout the Legionella genome. The mini-Tn10 insertions were stable during extracellular and intracellular growth, and did not alter the infectivity of L. pneumophila. Thus, this mutagenesis system offers an easy, one-step approach toward isolating large populations of random mutants which can be screened for defects in virulence.