SummaryLegionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dottranslocated substrates and several C. burnetii icm/ dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA-and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.Bacterial pathogens, as well as bacteria that live in close contact with eukaryotic cells, have developed many mechanisms to subvert their host cells and grow in intimate association with them. Many bacterial pathogens, such as Yersinia spp., Salmonella enterica, Pseudomonas aeroginosa, and Escherichia coli O157, use the type III secretion system as part of their pathogenesis determinants (14). Other bacteria such as Agrobacterium tumefaciens, Bordetella pertussis, and Legionella pneumophila use type IV secretion systems, which are functionally homologous to type III secretion systems but are evolutionarily related to bacterial conjugation systems, as opposed to the type III secretion systems, which are evolutionarily related to the bacterial flagellar basal body (9, 13).L. pneumophila, the causative agent of Legionnaires' disease, is a facultatively intracellular pathogen that is able to infect, multiply within, and kill human macrophages, as well as free-living amoebae (32, 48). Two regions of icm/dot genes that constitute the L. pneumophila icm/dot type IV secretion system have been discovered (reviewed in references 53 and 64). Region I contains 7 genes (icmV, -W, and -X and dotA, -B, -C, and -D) (3,6,39,63), and region II has been shown to contain 17 genes (icmT, -S, -R, -Q, -P, -O, -N, -M, -L, -K, -E, -G, -C, -D, -J, -B, and -F) (1,46,50,52,63). The icm/dot genes were shown to participate in m...

The signaling pathways leading to growth and patterning of various organs are tightly controlled during the development of any organism. These control mechanisms usually involve the utilization of feedbackand pathway-specific antagonists where the pathway induces the expression of its own antagonist. Sef is a feedback antagonist of fibroblast growth factor (FGF) signaling, which has been identified recently in zebrafish and mammals. Here, we report the isolation of chicken Sef (cSef) and demonstrate the conserved nature of the regulatory relationship with FGF signaling. In chick embryos, Sef is expressed in a pattern that coincides with many known sites of FGF signaling. In the developing limb, cSef is expressed in the mesoderm underlying the apical ectodermal ridge (AER) in the region known as the progress zone. cSef message first appeared after limb budding and AER formation. Expression was intense at stages of rapid limb outgrowth, and gradually decreased to almost undetectable levels when differentiation was clearly apparent. Gain-and loss-of-function experiments showed that FGFs differentially regulate the expression of cSef in various tissues. Thus, removal of the AER down-regulated cSef expression, and FGF2 but not

Cell fate determination is governed by complex signaling molecules at appropriate concentrations that regulate the cell decisionmaking process. In vertebrates, however, concentration and kinetic parameters are practically unknown, and therefore the mechanism by which these molecules interact is obscure. In myogenesis, for example, multipotent cells differentiate into skeletal muscle as a result of appropriate interplay between several signaling molecules, which is not sufficiently characterized. Here we demonstrate that treatment of biochemical events with SAT (satisfiability) formalism, which has been primarily applied for solving decision-making problems, can provide a simple conceptual tool for describing the relationship between causes and effects in biological phenomena. Specifically, we applied the Łukasiewicz logic to a diffusible protein system that leads to myogenesis. The creation of an automaton that describes the myogenesis SAT problem has led to a comprehensive overview of this non-trivial phenomenon and also to a hypothesis that was subsequently verified experimentally. This example demonstrates the power of applying Łukasiewicz logic in describing and predicting any decision-making problem in general, and developmental processes in particular.