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Listeria monocytogenes secretes two chitinases and one chitin binding protein. Mutants lacking chiA, chiB, or lmo2467 exhibited normal growth in cultured cells but were defective for growth in the livers and spleens of mice. Mammals lack chitin; thus, L. monocytogenes may have adapted chitinases to recognize alternative substrates to enhance pathogenesis.

Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17 degrees C and aquatic amoebae at 22 to 25 degrees C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.

Several Legionella pneumophila proteins were highly expressed in low-temperature supernatants. One of these proteins was the peptidyl-prolyl isomerase PpiB. Mutants lacking ppiB exhibited reduced growth at 17 degrees C. Since PpiB lacked a signal sequence and was present in 17 degrees C supernatants of type II and type IV secretion mutants, this protein may be secreted by a novel mechanism.

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.